Báo cáo khoa học: "Oak chloroplast-DNA polymorphisms detected by restriction fragment length polymorphism (RFLP)"

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Note Oak chloroplast-DNA polymorphisms detected by restriction fragment length polymorphism (RFLP) J Glössl J Schmidt 2 M Zechmeister-Machhart K Burg 1 Austrian Research Center, Department of Biotechnology, Seibersdorf; 2 Center of Applied Genetics, University of Bodenkultur, Vienna, Austria Summary — Petunia hybrida chloroplast (cp) DNA probes were used to find restriction fragment length polymorphisms (RFLPs) in the cp DNA of the oak species Quercus robur and Quercus pe- traea. Five individuals have been analysed (2 Q robur and 3 Q petraea) with 18 different restriction enzymes, and with 7 P hybrida cp DNA probes. Only 2 probes (P6 and P8) detected polymorphisms, probe P6 detected 4 polymorphisms with the restriction enzymes Aval, BglII, Clal and Xbal, while probe P8 detected a BglII polymorphism. fragment length polymorphism (RFLP) oak / chloroplast / restriction Polymorphisme de longueur des fragments de restriction de l’ADN chloroplas- Résumé — les chênes. Des sondes du génome chloroplastique de Petunia hybrida ont été utilisées tique chez pour la recherche de polymorphisme de longueur de fragment de restriction dans l’ADN chloroplas- tique (cp) de Quercus robur et Quercus petraea. L’ADNcp de 5 arbres (2 Q robur et 3 Q petraea) a été digéré par 18 enzymes de restriction et hybridé avec 7 sondes de P hybrida. Deux sondes seule- ment ont révélé du polymorphisme (P6 et P8) : P6 avec les enzymes de restriction Aval, BglII, ClaI et XbaI; P8 avec l’enzyme BglII. / chloroplaste / polymorphisme de longueur de fragment de restriction (RFLP) Quercus turn diminish the capacity of populations INTRODUCTION to respond to new selective pressures. we need better insight into the Therefore, Climatic variations as well as human activ- and ecology of populations in or- genetics ities (eg environmental pollution) greatly der to minimize the negative effects of hu- influence natural ecosystems, frequently activity. man restricting the habitat, reproductive poten- Genetic markers needed to under- are tial and number of individuals of many population’s genetic events tak- stand the species. ing place in forest tree species, eg, to These restrictions may reduce genetic study inheritance patterns, however, the which may in numbers of available genetic markers are variability within populations,
In our experiments, we used a Petunia hybri- very limited in forest tree species. In partic- da cpDNA library (originally described by Palmer ular, little information is available on the et al, 1983), kindly provided by Dr D Neale, to oak pecies Quercus robur and Quercus identify oak cpDNA polymorphisms. DNA probes petraea, which are the focus of our inter- were labeled with [α- by random prim- P]dATP 32 est, as the 2 most important oak species in ing (Boehringer-Mannheim). Filter hybridization Austria. was performed as described by Church and Gil- bert (1984), at 50°C overnight. Three washes Direct DNA analysis by restriction frag- were made in 2 x SSC, 0.1 % (w/v) sodium dode- ment length polymorphism (RFLP), pro- cyl sulfate (SDS) at room temperature (5 min duces a theoretically infinite number of each), followed by 1 x SSC, 0.1% SDS washes DNA markers. These markers can be cho- at 50°C for 30 min. The wet filters were exposed sen from coding or non-coding regions of to Kodak X-Omat autoradiographic films with 2 nuclear and cytoplasmic genomes (chloro- intensifying screens (DuPont) at -80°C. plast and mitochondrial). As far as oak species are concerned, RESULTS few RFLP data are available (ie, Bellarosa, 1990; Petit et al, 1990; Whittemore and Schaal, 1991). Since cpDNA sequences are rather con- However, development of probes de- servative, it was possible to use heterolo- tecting polymorphisms in the chloroplast gous petunia cpDNA probes. (cp) DNA of oak species, would enable us During this study we analyzed 5 oak to follow the inheritance of the most con- individuals (2 Q robur and 3 Q petraea) servative DNA sequences of plants. with 18 different restriction enzymes (ta- cpDNA polymorphisms could provide infor- ble I) and with 7 different P hybrida cpDNA mation on evolutionary distances among clones (table I). RFLPs were found only oak species, the origins of populations and with the probes P6 and P8. Hybridizations the occurrence of interspecies crosses. with the probe P6 revealed several poly- In this paper, we report on DNA probes morphisms. In the case of the enzyme which detect RFLP variation in Q robur Aval this probe detected 8 constant frag- and Q petraea. ments in both species (12.5, 7.6, 4.7, 3.8, 2.3, 2.0, 1.8 and 1.65 kilobases (kb) in size). Additionally, in Q robur samples there MATERIALS AND METHODS were 2 fragments (3.0 and 1.0 kb) which were not present in Q petraea samples. In- Leaf material of both Quercus robur and Quer- stead there was a 7.0-kb fragment and in petraea was collected in the southeastern cus Q petraea samples (fig 1A). The same part of province Burgenland in the forest domain probe revealed 3 common BglII fragments Prince of Bavaria in Austria. (11.0, 3.3 and 1.1kb in size) and a vari- DNA was extracted as described previously able one which of either 3.6 or 5.0 kb in Kreike et al (1991). DNA samples of 1 μg by the Q robur and Q petraea samples, re- were digested by Boehringer-Mannheim restric- spectively (fig 1B). In the case of Xbal di- tion endonucleases according to the manufac- gestion, 6 constant fragments were found turer’s recommendations. The digested samples were loaded onto 0.8% agarose gels (Sigma, A- (17, 6.5, 3.1, 2.8, 1.4 and 1.25 kb) and a 9539) and run overnight at approximately 1 V/ 9.0-kb band was also present in the Q pe- cm. Alkaline capillary blotting to Hybond N filters traea samples (fig 1 D). Four constant Clal (Amersham) was done according to the manu- fragments were found (3.5, 3.2, 3.0 and facturer’s recommendations. DNA was fixed on 1.8 kb), while a 2.5-kb restriction fragment the filter by heating at 80°C for 2 h.
of the fragments adja- Probe P8, one cent to P6 in Petunia, detected a BglII frag- ment similar to that of P6 in Q petraea samples (5-kb fragment). However, the smaller polymorphic fragment found in Q robur samples (3.6-kb fragment) was not detected (fig 1 C). CONCLUSIONS we described 5 poly- In the present report, sites within the oak cpDNA. Four morphic of these 5 polymorphisms were detected by the Petunia cpDNA probe P6, while the 5th one was identified by the neighboring Petunia probe P8. The other Petunia cpDNA probes did not show polymorphisms with the restriction enzymes used. The polymorphism detected by the P6 Petunia only in the Q petraea sam- detected was probe with BglII had been described earlier In the latter two cases, how- ples (fig 1 E). (Kreike et al, 1991). The other poly- ever, the hybridization signal of the poly- morphisms are new ones not reported to morphic fragment was weaker than that of date. the constant ones.
ber 1990. Göttingen, Germany (Müller-Starck REFERENCES G, Ziehe M, eds) JD Sauerländer-Verlag Palmer JD, Shields CR, Cohen DB, Orton TJ R, Delre V, Schirone B, Maggini F Bellarosa (1983) Chloroplast DNA evolution and the or- (1990) Ribosomal RNA genes in Quercus igin of amphidiploid Brassica species. Theor ssp (Fagaceae). Plant Syst Evol 172, 127- Appl Genet 65, 181-189 139 Petit RJ, Wagner DB, Kremer A (1990) An effi- Church GM, Gilbert W (1984) Genomic se- cient method for chloroplast DNA surveys: quencing. Proc Natl Acad Sci USA 81, 1991- application to Quercus species in western 1994 Europe. Int Symp Population Genetics of For- Kreike J, Burg K, Zechmeister M, Haider T, est Trees, July 31-August 2. Oregon State Glössl J (1991) DNA-fingerprint and RFLP University, Corvallis, OR, USA (abstr) analysis as tools to study genetic diversity in populations of fir, spruce and oak. In: Proc Whittemore AT, Schaal BA (1991) Interspecific EEC Workshop on Genetic Variation of For- gene flow in sympatric oaks. Proc Natl Acad est Tree Populations in Europe. 9-11 Octo- Sci USA 88, 2540-2544